+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23282 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | polynucleotide phosphorylase | |||||||||
Map data | ||||||||||
Sample |
| |||||||||
Keywords | phosphorylase polymerase RNA decay / RNA BINDING PROTEIN / TRANSFERASE-RNA complex | |||||||||
Function / homology | Function and homology information polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mRNA catabolic process / RNA processing / magnesium ion binding / RNA binding / cytoplasm Similarity search - Function | |||||||||
Biological species | Mycolicibacterium smegmatis (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.07 Å | |||||||||
Authors | Goldgur Y / Shuman S | |||||||||
Funding support | United States, 1 items
| |||||||||
Citation | Journal: RNA / Year: 2021 Title: Structure and mechanism of polynucleotide phosphorylase. Authors: Mihaela-Carmen Unciuleac / Shreya Ghosh / M Jason de la Cruz / Yehuda Goldgur / Stewart Shuman Abstract: Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts ...Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase. | |||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23282.map.gz | 97 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-23282-v30.xml emd-23282.xml | 11.3 KB 11.3 KB | Display Display | EMDB header |
Images | emd_23282.png | 71.1 KB | ||
Filedesc metadata | emd-23282.cif.gz | 5.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23282 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23282 | HTTPS FTP |
-Validation report
Summary document | emd_23282_validation.pdf.gz | 478.9 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_23282_full_validation.pdf.gz | 478.5 KB | Display | |
Data in XML | emd_23282_validation.xml.gz | 6.6 KB | Display | |
Data in CIF | emd_23282_validation.cif.gz | 7.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23282 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23282 | HTTPS FTP |
-Related structure data
Related structure data | 7ld5MC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_23282.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Voxel size | X=Y=Z: 1.064 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : Polynucleotide phosphorylase complex in poly(A) synthesis
Entire | Name: Polynucleotide phosphorylase complex in poly(A) synthesis |
---|---|
Components |
|
-Supramolecule #1: Polynucleotide phosphorylase complex in poly(A) synthesis
Supramolecule | Name: Polynucleotide phosphorylase complex in poly(A) synthesis type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
---|---|
Source (natural) | Organism: Mycolicibacterium smegmatis (bacteria) |
-Macromolecule #1: Polyribonucleotide nucleotidyltransferase
Macromolecule | Name: Polyribonucleotide nucleotidyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: polyribonucleotide nucleotidyltransferase |
---|---|
Source (natural) | Organism: Mycolicibacterium smegmatis (bacteria) |
Molecular weight | Theoretical: 83.647703 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGHHHHHHHH HHSSGHIEGR HMSVVELEDG VYESTAVIDN GSFGTRTIRF ETGRLAQQAA GSAVAYLDDE TMLLSATTAS KNPKDHFDF FPLTVDVEER MYAAGRIPGS FFRREGRPST DAILTCRLID RPLRPSFVDG LRNEIQVVVT VMSLDPKDLY D VLAINAAS ...String: MGHHHHHHHH HHSSGHIEGR HMSVVELEDG VYESTAVIDN GSFGTRTIRF ETGRLAQQAA GSAVAYLDDE TMLLSATTAS KNPKDHFDF FPLTVDVEER MYAAGRIPGS FFRREGRPST DAILTCRLID RPLRPSFVDG LRNEIQVVVT VMSLDPKDLY D VLAINAAS MSTQLAGLPF SGPVGGARIA LIDGTWVAFP TVEQLERAVF DMVVAGRIVG DGDSADVAIM MVEAEATENV VE LVAGGAQ APTEAVVAEG LEAAKPFIKA LCAAQQELAD RAAKPAGEYP VFPDYEADVY DAVASVATEA LAEALTIAGK TER NDRTDE IKVEVLERLA EPYAGREKEI GAAFRSLTKK LVRQRILTDH FRIDGRGITD IRALSAEVAV IPRAHGSALF ERGE TQILG VTTLDMIKMA QQIDSLGPEN TKRYMHHYNF PPYSTGETGR VGSPKRREIG HGALAERALV PVLPSIEEFP YAIRQ VSEA LGSNGSTSMG SVCASTLALL NAGVPLKAPV AGIAMGLVSD DVDVDGKVEK RYVALTDILG AEDAFGDMDF KVAGTK DFV TALQLDTKLD GIPSQVLAGA LSQAKDARLT ILDVMAEAID RPDEMSPYAP RITTIKVPVD KIGEVIGPKG KMINSIT EE TGAQISIEDD GTVFVGAADG LSAQAAIDKI NAIANPQLPK VGERFLGTVV KTTDFGAFVS LLPGRDGLVH ISKLGKGK R IAKVEDVVKV GDKLRVEIAD IDNRGKISLV LVAEESAESA ESAGDKGAEK AEGAAADVTP AEA UniProtKB: Polyribonucleotide nucleotidyltransferase |
-Macromolecule #2: poly-A RNA fragment
Macromolecule | Name: poly-A RNA fragment / type: rna / ID: 2 / Number of copies: 3 |
---|---|
Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 2.917895 KDa |
Sequence | String: AAAAAAAAA |
-Macromolecule #3: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: MG |
---|---|
Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
---|---|
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: INSILICO MODEL / In silico model: via cryoSPARC 3D ab-initio model |
---|---|
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 890249 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.16.1) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.16.1) |
-Atomic model buiding 1
Refinement | Space: REAL |
---|---|
Output model | PDB-7ld5: |